Influenza virus binding, sialylated oligosaccharide substance and use thereof

ABSTRACT

The present invention is directed to human influenza virus binding substance containing at least one oligosaccharide chain, which comprises a terminal NeuNAcα6 linked to: (a) a linear or branched polylactosamine type structure consisting of at least three lactosamine residues, a linear sequence optionally containing one or two α3-linked fucose residues in a non-sialylated lactosamine, a branched structure optionally carrying one or more additional NeuNAcα-residues at a terminal position in a branch, and/or (b) a linear or branched structure with two lactosamine and one lactose residue, a linear structure in addition containing one or two α3-linked fucose residues in a non-sialylated lactosamine or lactose, a branched structure optionally carrying one additional NeuNAcα-residue in a terminal position of the branch, or an analog or derivative of said oligosaccharide chain for use in binding of human influenza virus.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/F101/00587 which has an Internationalfiling date of Jun. 20, 2001, which designated the United States ofAmerica.

FIELD OF THE INVENTION

The present invention relates to a substance or a receptor binding tohuman influenza virus, as well as use thereof in pharmaceuticalcompositions and a method for treatment of a condition due to thepresence of influenza virus in the human respiratory tract. Theinvention is also directed to the use of the receptor for diagnostics ofinfluenza viruses.

BACKGROUND OF THE INVENTION

Influenza virus attachment to host cells is mediated by specificinteractions of the viral envelope protein hemagglutinin (HA) withsialylated carbohydrate chains of cell surface glycoproteins andglycolipids (for reviews, Suzuki, 1994; Herrler et al., 1995; Paulson,1985; Wiley and Skehel, 1987). Natural sialylglycoproteins andgangliosides exhibit significant structural diversity, and differentreceptors are probably utilized by the viruses in different hosttissues. It has been shown, that influenza A viruses isolated from avianspecies preferentially bind to NeuAcα3Gal-terminated sugar chains, whileclosely related human viruses reveal a higher binding affinity towardsthe NeuAcα6Gal-terminated structures (Paulson, 1985; Suzuki, 1994;Connor et al., 1994; Matrosovich et al., 1997; Gambaryan et al, 1997).Additional influential features for binding are inner parts ofsaccharide chains (Gambaryan et al., 1995; Matrosovich et al., 1997;Rogers and Paulson, 1983; Suzuki et al., 1987; Suzuki et al., 1992;Eisen et al., 1997), polyvalency of receptor saccharides (Pritchett andPaulson, 1989; Matrosovich, 1989; Mammen et al., 1995), spatialarrangement of sialyloligosaccharides in receptor glycoproteins(Pritchett and Paulson, 1989), or glycosylation of the viralhemagglutinins (Gambaryan et al., 1998; Ohuchi et al., 1997). Detailedmolecular mechanisms of these effects and the importance of a variationin fine structure of sialylated receptors for the virulence andpathogenicity of individual viral strains are not known. For examplevirulence of the 1918 influenza pandemic still remains unexplained(Laver et al., 1999), and the actual strains have not been assayed forreceptor specificity.

Studies on the structural characterization of biological receptors forhuman influenza viruses are hampered by the limited availability of thehuman respiratory tract tissues. However, characterization of thebinding molecules from other human tissues may permit furtherspecification of the receptor binding epitopes. Human leukocytesrepresent an attractive experimental model because they contain a seriesof gangliosides with high binding affinity for the virus. Bindingspecies were detected in human leukocytes among common gangliosides(Müthing et al. 1993; Müthing, 1996). In contrast to the presentinvention these were described to contain α3-sialylated sialyl-Lewis xand VIM-2 sequences. The receptor activity was also observed amonghighly complex glycolipid fractions, polyglycosylceramides (Matrosovichet al., 1996). NeuAcα6-containing glycolipids of human leukocytes withmore than two lactosamine units in the core chain have not yet beencharacterized by other laboratories (Müthing et al., 1993; Müthing etal. 1996; Müthing, 1996; Stroud et al., 1995; Stroud et al., 1996). Theyoccur in human white cells in very small amounts and their existence hasso far been neglected. However, these minor species may be of biologicalimportance for in vivo events during influenza infections and mayexplain virulence variations between strains (Laver et al., 1999).

Binding of influenza viruses to sialic acid-containing neutrophilreceptor(s) depresses bactericidal activity of neutrophils (Abramson andMills, 1988; Cassidy et al., 1989; Daigneault et al., 1992; Abramson andHudnor, 1995) and stimulates apoptosis of these cells by a yet undefinedmechanism (Colamussi et al., 1999). This virus-mediated neutrophildysfunction is a likely contributor to the development of secondarybacterial infections, which are the main cause of morbidity andmortality during influenza epidemics.

Several studies describe sialylated di- to heptasaccharide bindingstructures for human influenza viruses e.g. (Gambaryan et al., 1995;Matrosovich et al., 1997; Rogers and Paulson, 1983; Suzuki et al., 1992;Eisen et al., 1997). These have low affinities to the viruses and thestudies do not describe larger saccharide receptors which have the highnatural binding affinity. The divalent NeuNAcα6Galβ4GlcNAc-saccharidesconstructed chemically on β-galactosides are described in Sabesan, S. etal. -92 and in U.S. Pat. Nos. 5,254,676 and 5,220,008 and have onlymodestly better affinities than corresponding monosialocompounds evenwhen linked on bovine serum albumin in multivalent form.

Several synthetic polymeric influenza inhibitors containing sialic acid(Mammen et al., 1995) or sialylated lactose/N-acetyllactosamine havebeen described (Gambaryan et al, 1997). The specific binding of humaninfluenza virus to NeuNAcα6Galβ4GlcNAc-epitopes (Gambaryan et al, 1997)and the presence of NeuNAcα6Gal on the surface of human-ciliatedtracheal epithelium (Baum and Paulson, 1990) were suggested toconstitute an essential part of the biologically relevant cellularreceptor for the viruses (Gambaryan et al, 1997).

A polylactosamine containing and α6-sialylated inhibitor of humaninfluenza viruses has been described. This molecule is produced from acryptically I-active (anti-I-antibodies are known to recognizepolylactosamine and non-polylactosamine structures) glycoprotein 2 ofbovine erythrocytes by removing sialic acid residues by sialidasetreatment and by enzymatic resialylation by α3- or α6-sialyltransferases(Suzuki et al., 1987). The semisynthetic bovine protein is not a naturalreceptor structure of human viruses. It is hardly useful as atherapeutic inhibitor of human influenza viruses, because it contains aprotein structure and a substantial amount ofGalα3Galβ4GlcNAc-xeno-antigenic structures (Suzuki et al, 1985). TheGalα3Galβ4GlcNAc-antigen is present in many mammalian species but notproduced by human tissues, the structure is highly antigenic in human,and there are naturally large amounts of antibodies against thestructure in humans. Foreign protein structures are known to bepotential antigens and allergens for humans.

The authors (Suzuki et al., 1987) discuss that inner I-activeneolacto-series type II sugar chains may also be important as a commonpart of the receptor determinant toward the hemagglutinin of humaninfluenza viruses A and B. Unfortunately their saccharide material isvery heterogenous and the possible sialylated I-active components formedwere not characterized chemically and no specific epitopes were assignedas virus binding structures. According to the data of FIG. 3corresponding to oligosaccharides labelled at the reducing end, thelarge I-polylactosamines are actually very minor species in theglycoprotein II and smaller O-glycans are major species in molar amount,the smaller O-glycans without branched polylactosamime are alsorecognized by at least one anti-I-antibody used to determine thesaccharides of the protein (Suzuki et al., 1985). In another study thesame authors show that α3-sialylated and α3-galactosylated branchedpolylactosamine glycolipid (also an I-antigenic structure) was shown tobe a binding compound for influenza virus (Suzuki et al., 1986).

A BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Binding of HRP conjugates of human (H1N1) and avian (H4N6)influenza A viruses to gangliosides isolated from different humantissues and separated on silica gel thin-layer plates. Anis, platesprayed with anisaldehyde (4-methoxybenzaldehyde); Human virus and Avianvirus, plates overlayed with respective conjugates. Lane 1, upper phasegangliosides (after Folch's partition) from human leukocytes; Lane 2,upper phase gangliosides from human erythrocytes; Lane 3, totalgangliosides from human small intestine, sample 1; Lane 4, totalgangliosides from human small intestine, sample 2; Lane 5, totalgangliosides from human stomach; Lane 6, total gangliosides from humanmeconium; Lane 7, total gangliosides from human colon; Lane 3, totalgangliosides from human pancreas; Lane 9, bovine brain gangliosides. Theplates were developed in Chloroform/Methanol/0.25% KCl in H₂O, 50:40:10.S-3-PG, sialyl-3-paragloboside; S-6-PG, sialyl-6-paragloboside; 5s, 7sand 8s, 5-, 7- and 8-sugar-containing monosialoganglioside fractions(NeuAc₁Hex₃HexNAc₁Cer, NeuAc₁Hex₄HexNAc₂Cer andFuc₁NeuAc₁Hex₄HexNAc₂Cer, respectively). To visualize slowly migratinggangliosides some lanes were overloaded. Dotted lines were drawn tofacilitate interpretation.

FIG. 2. Binding of human and avian influenza viruses HRP conjugates toleukocyte gangliosides on silica gel TLC plates before (lane 1) andafter (lane 2) mild oxidation and reduction. Lane 3: reference braingangliosides (from top: GM1, GD1a, GD1b, GT1b). Anis, gangliosidesstained with anisaldehyde. Chromatographic conditions were as in FIG. 1.x, non-sugar spot acquired during dialysis.

FIG. 3A. Negative ion FAB mass spectra of gangliosides from humanleukocytes before mild periodate oxidation and reduction.

FIG. 3B. Negative ion FAB mass spectra of gangliosides from humanleukocytes after mild periodate oxidation and reduction.

FIG. 4A. Binding of anti-sialyl-Lewis x (SLX) and Vim-2 (VIM) monoclonalantibodies to upper phase gangliosides (after Folch's partition) ofhuman leukocytes separated on silica gel TLC plates. Anis, gangliosidesstained with anisaldehyde; Virus, gangliosides overlaid with humaninfluenza virus BRP conjugate. The plates were developed inChloroform/Methanol/0.25% KCl, 50:55:13.

FIG. 4B. Binding of anti-sialyl-Lewis x (SLX) and Vim-2 (VIM) monoclonalantibodies to upper phase gangliosides (after Folch's partition) ofhuman leukocytes separated on silica gel TLC plates. Anis, gangliosidesstained with anisaldehyde; Virus, gangliosides overlaid with humaninfluenza virus HRP conjugate. The plates were developed inChloroform/Methanol/0.25% KCl, 50:40:10.

FIG. 5. Binding of NeuAcα3- and NeuAcα6-specific lectins from Maackiaamurensis (MAA) and Sambucus nigra (SNA) to gangliosides separated byTLC and blotted to PVDF membranes; Human virus, binding of human virusHRP conjugate on the corresponding TLC plate; Anis, TLC plate withseparated gangliosides and visualized with anisaldehyde. The plates weredeveloped in Chloroform/Methanol/0.250% KCl, 50:50:13. Lane 1, upperphase gangliosides (after Folch's partition) from human leukocytes; Lane2, sialyl-3-paragloboside; Lane 3, bovine brain gangliosides (GM1, GD1a,GD1b and GT1b).

FIG. 6. Binding of HRP conjugate of human (A, H1N1) influenza virus toganglioside subflactions (Fr. 1-6) obtained from a mixture of humanleukocyte gangliosides after preparative TLC; TG, Total gangliosidemixture. Anis, plate visualized with anisaldehyde; Human virus, plateoverlaid with virus conjugate.

FIG. 7. MALDI TOF spectrum of ganglioside fraction 6 of FIG. 6. Massspectrometer was operated in a negative-ion reflectron mode.

DETAILED DESCRIPTION OF THE INVENTION

In the invention, the binding of human viruses to gangliosides fromdifferent human tissues and cells, including leukocytes, was studied.The human virus bound selectively to only minor extended gangliosidespecies. So far other laboratories have not been able to detect andisolate the minor active species containing NeuNAcα6 sequences even whenstarting with huge amounts of leukocytes. The combination of the olderand newly developed special overlay techniques used by the inventorstogether with the most sensitive mass spectrometry experiments allowedanalysis of the novel receptors for human influenza virus.

Sialyl-Lewis x and VIM-2-active saccharides were excluded as parts ofthe active receptors binding the human influenza virus. Human is knownnot to contain Galα3-structures Larsen et al, 1990). It was also shownusing SNA lectin that the binding was to some minor NeuNAcα6-containingspecies. In addition, it was demonstrated, that the binding wasdependent on the presence of the unchanged glycerol sialic acid tail.The monosaccharide and ceramide compositions of the smallest activemolecules were indicated by mass spectrometric data of the activefraction. Mass spectrometry data together with the other evidenceallowed us to describe further potential structural features present inthe high affinity species. Assignments are based on the knowledge thatthe HexNAc_(x)Hex_(x+2) in the glycolipid fractions correspond topolylactosamine sequences. The structural features described are inaccordance with the previously known glycolipid structures of humanleukocytes (Stroud et al., 1995; Stroud et al., 1996; Stroud et al.,1996b; Müthing, 1996, Johansson and Miller-Podraza, 1998) except for thepresence α6-linked sialic acid on the structures.

The invention first describes the minimum structural elements present inthe high affinity receptor of human influenza virus. The receptor activespecies contain at least one oligosaccharide chain, which comprises aterminal NeuNAcα6 linked to

-   -   (a) -a linear or branched polylactosamine type structure        consisting of at least three lactosamine residues, a linear        sequence optionally containing one or two α3-linked fucose        residues in a non-sialylated lactosamine, a branched structure        optionally carrying one or more additional NeuNAcα-residues at a        terminal position in a branch, and/or    -   (b) -a linear or branched structure with two lactosamine and one        lactose residue, a linear structure in addition containing one        or two α3-linked fucose residues in a non-sialylated        lactoseamine or lactose, a branched structure optionally        carrying one additional NeuNAcα-residue in a terminal position        of the branch. These active receptor epitopes are called virus        binding structures.

The virus binding substances contain no Galα3Galβ4GlcNAc-residues asactive virus binding parts and is preferably not linked to a protein andpreferably do not contain any Galα3Galβ4GlcNAc-xenoantigenic structures.The virus binding substances are preferentially presented in clusteredform such as by glycolipids on cell membranes, micelles, liposomes or onsolid phases such as TCL-plates used in the assays. The clusteredrepresentation with correct spacing creates high affinity binding.According to a preferred embodiment the virus binding substance thuscontains at least two oligosaccharides as defined, preferably at leastthree or at least four oligosaccharides as defined. It is noted from theTLC-assays that larger polylactosamine type compounds containing theNeuNAcα6-terminal are also receptor for the viruses. These compoundspresent in even more minor amounts are very high affinity receptors forthe virus. Repeating virus binding epitopes containing branches withNeuNAcα6Galβ4GlcNAc on larger polylactosamine structures can effectivelybind to several binding sites of virus surfaces, most probably on thehemagglutinin protein. The large glycolipid fraction is continuous withlarger molecular weight polylactosamine glycolipid fraction calledpolyglycosylceramides. It has been recently shown that thepolyglycosylceramide fraction has very high affinity for humaninfluenza, virus (Matrosovich et. al, 1996) and in a separate studythese polylactosamines have been shown to carry NeuNAcα6-terminalstructures (Johansson and Miller-Podraza, 1998). In a preferredembodiment the virus binding substances are presented onpolylactosamine-type oligo- or polysaccharide or glycosides thereof. Inthe middle and nonreducing terminal parts of natural virus bindingpolylactosamines the binding epitope does not contain lactose residues,but a lactose residue may be present at the reducing end.

According to the invention it is possible to use the influenza virusbinding epitopes or naturally occurring or a synthetically producedanalogue or derivative thereof having a similar or better bindingactivity with regards to human influenza virus. It is also possible touse a substance containing the virus binding substance, such as areceptor active polylactosamine ganglioside described in the inventionor an analogue or derivative thereof having a similar or better bindingactivity with regards to human influenza virus.

The virus binding substance may be a glycosidically linked terminalepitope of an oligosaccharide chain. Alternatively the virus bindingepitope may be a branch of a polylactosamine chain.

Further assignment of the most potential receptor structures is possiblebased on the following facts. The SNA-lectin recognizes the terminalNeuNAcα6Gal-sequence. The presence of two NeuNAcs with α6 or α3-linkagesindicates that the N-acetyllactosamine structures are branched. Thebiosynthetic knowledge in the art indicates that terminalNeuNAcα6Galβ4GlcNAc sequences are not α3 fucosylated (e.g. Paulson etal, 1978; DeVries et al, 1995), indicating that fucose residues arelocated further away from terminal NeuNAc as inNeuNAcα6Galβ4GlcNAcβ3Galβ4(Fucα3)GlcNAcβ. Branching and fucosylation areexclusive on the same N-acetyllactosamine sequence of the knownleucocyte type enzymes (Niemelä et al., 1998; Mattila et al., 1998).Furthermore, the Glc-residue is not fucosylated by the majorfucosyltransferase of leukocytes (De Vries et al., 1995; Clarke andWatkins, 1996), which is in accordance with the knowledge of the vastmajority of the isolated human oligosaccharide structures. However, theanalysis does not exclude the presence of other minor isomeric specieswhich are synthesized by yet uncharacterized biosynthetic reactions.Under the assay conditions described, the linear chain glycolipidsNeuNAcα6Galα4GlcNAcβ3Galβ4GlcβCer orNeuNAcα6Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4GlcβCer did not show binding,indicating specific structural features on the larger bindingglycolipids.

Mass spectrometry indicated the presence of disialylated and/ordifucosylated molecules in the active fraction with an abundance of theactive species, disialylation and difucosylation being structuralfeatures different from the non-active linear sequences. Thedisialylated species matches most correctly to the molecular masses ofthe saccharide sequences with the same lipid species previously known tobe present in major sialylated glycolipids of leukocytes. These dataindicate that the disialylated and/or difucosylated species belong tothe high affinity receptor species.

The saccharide sequences of the receptor disialylated molecules areaccording to the experimental evidence and current biosynthetic andstructural knowledge: NeuNAcα6Galβ4GlcNAcβ3(NeuNAcα6/or3Galβ4GlcNAcβ6)Galβ4GlcNAcβ-3Galβ4GlcβCer andNeuNAcα6Galβ4GlcNAcβ3(NeuNAcα6/or 3Galβ4GlcNAcβ6)Galβ-4GlcβCer. Thelectin binding assays indicate that most of the active species aredevoid of NeuNAcα3, therefore indicating species having both brancheswith NeuNAcα6:NeuNAcα6Galβ4GlcNAcβ3(NeuNAcα6Galβ4GlcNAcβ6)Galβ4GlcNAcβ3Galβ4GlcβCerand NeuNAcα6Galβ4GlcNAcβ3(NeuNAcα6Galβ4GlcNAcβ6)Galβ4GlcβCer. Thebinding of the more elongated decasaccharide sequences to the influenzavirus may be more effective. The similar terminal octasaccharidesequences are defined as virus binding substances. The preferreddisialylated virus binding substances have the structures:NeuNAcα6Galβ4GlcNAcβ3(NeuNAcα6/or 3Galβ4GlcNAcβ6)Galβ4GlcNAc,NeuNAcα6Galβ4GlcNAcβ3(NeuNAcα6/or 3Galβ4GlcNAcβ6)Galβ4Glc,NeuNAcα6Galβ4GlcNAcβ3(NeuNAcα6Galβ4GlcNAcβ6)Galβ4GlcNAc, andNeuNAcα6Galβ4GlcNAcβ3(NeuNAcα6Galβ4GlcNAcβ6)Galβ4Glc.

The saccharide sequences of the receptor difucosylated molecules areaccording to the experimental evidence and current biosynthetic andstructural knowledge:NeuNAcα6Galβ4GlcNAcβ3Galβ4(Fucα3)GlcNAcβ3Galβ4(Fucα3)GlcNAcβ3Galβ4Glcβ-Cerand a less likely form of fucosylated lactose:NeuNAcα6Galβ4GlcNAcβ3Galβ4(Fucα3)GlcNAcβ3Galβ4(Fucα3)GlcβCer, thesaccharide sequence of this is considered as an effective analog of theterminal nonasaccharide sequence of the longer version. The specificstructural feature of difucosylation together with NeuNAcα6 isconsidered to be active part in the recognition of the virus.Preferentially the difucosylated virus binding structures areNeuNAcα6Galβ4GlcNAcβ3Galβ4(Fucα3)GlcNAcβ3Galβ4(Fucα3)GlcNAc andNeuNAcα6Galβ4GlcNAcβ3Galβ4(Fucα3)GlcNAcβ3Galβ4(Fucα3)Glc.

A further extension of the glycolipid could be a structural featureincreasing the receptor activity in comparison to the non-activespecies. Mass spectrometry peaks indicating the presence of the majornon-receptor active α3-sialylated species, may also contain signalscorresponding to isomeric α6-sialylated molecules. The extendedreceptors have structuresNeuNAcα6Galβ4GlcNAcβ3Galβ4(Fucα3)₀₋₁GlcNAcβ3Galβ4(Fucα3)₀₋₁GlcNAcβ3Galβ4GlcβCer, where the molecule has 0 or 1 fucosyl residues.Furthermore a branched monosialylated structure is possible with thesame molecular weight as the linear non-sialylated forms such asNeuNAcα6Galβ4GlcNAcβ3(Galβ4GlcNAcβ6)-Galβ4GlcNAcβ3Galβ4GlcβCer. Theextended linear and branched structural features are very similar to thespecies indicated above and therefore these molecules are likely to alsohave receptor activity. The sequencesNeuNAcα6Galβ4GlcNAcβ3Galβ4(Fucα3)₀₋₁GlcNAcβ3Galβ4(Fucα3)₀₋₁GlcNAc, wherethe molecule has 0 or 1 fucosyl residues andNeuNAcα6Galβ4GlcNAcβ3(Galβ4GlcNAcα6)Galβ4GlcNAc are also virus bindingstructures.

When present as a branch of a longer polylactosamine chain the virusbinding substance has the structure R-3 or6Galβ4GlcNAcβ6/3(NeuNAcα6Galβ4GlcNAcβ3/6)Galβ-4GlcNAcβ4R₂, where the Ris a glycosidically linked non-reducing end part of the polylactosamine,and R₂ is glycosidically linked to the reducing end part of thepolylactosamine chain, preferably the R and/or R₂ contain more virusbinding substances. The reducing end of the saccharide may be furtherderivatized.

The oligosaccharide chain of the invention can be a part of apolylactosamine chain or a conjugate thereof. Preferably, thepolylactosamine chain contains at least 6 lactosamine residues or atleast three sialic acid residues.

The virus binding substances may be conjugated to a carrier. Theconjugation is performed by inking the virus binding substancepreferably from the reducing end to a carrier molecule. When theconjugate is used in therapeutics, the carrier molecule is preferablynot a protein.

The virus binding substances, preferably in oligovalent or clusteredform, can be used to treat a disease or condition caused by the presenceof the influenza virus in the respiratory tract of a patient. In suchcase the substances of the invention are used for anti-adhesion, i.e. toinhibit the binding of human influenza viruses to the receptor epitopesof the target cells or tissues. The target cells are neutrophils orepithelial cells of the respiratory tract. When the substance orpharmaceutical composition according to the invention is administered itwill compete with the receptor glycoconjugates on the target cells forthe binding of the viruses. Some or all of the viruses will then bebound to the substance according to the invention instead of thereceptor on the target cells or tissues. The viruses bound to thesubstances according to the invention are then removed from therespiratory tract with secreted mucous material, resulting in a reducedeffect of the viruses to the health of the patient. Preferably thepharmaceutical composition used is a soluble composition comprising thesubstances of the invention. The substance according to the inventioncan be attached to a carrier substance which is preferably not aprotein. When using a carrier molecule, several molecules of thesubstance according to the invention can be attached to one carrier andthe inhibitory efficiency is improved.

According to the invention it is possible to incorporate the substanceaccording to the invention, optionally with a carrier, in apharmaceutical composition, which is suitable for the treatment of acondition due to the presence of influenza virus in the respiratorytract of a patient or to use the substance according to the invention ina method for treatment of such conditions. Examples of conditionstreatable according to the invention are virus-mediated neutrophildysfunction which includes depression of bactericidal activity ofneutrophils and stimulation apoptosis of these cells, prevention ofsecondary bacterial infections due the neutrophil dysfunction, and theprimary infection by the influenza viruses in the respiratory tract.

The pharmaceutical composition according to the invention may alsocomprise other substances, such as an inert vehicle, or pharmaceuticallyacceptable carriers, preservatives etc. which are well known to personsskilled in the art.

The substance or pharmaceutical composition according to the inventionmay be administered in any suitable way, although an oral or nasaladministration especially in the form of a spray or inhalation arepreferred.

The term “treatment” used herein relates both to treatment in order tocure or alleviate a disease or a condition, and to treatment in order toprevent the development of a disease or a condition. The treatment maybe either performed in a acute or in a chronic way.

The term “patient”, as used herein, relates to any human or non-humanmammal in need of treatment according to the invention.

Furthermore, it is possible to use substances specifically binding orinactivating the substances according to the invention when present onhuman tissues and thus to prevent the binding of influenza virus.Examples of such substances include the lectin Sambucus nigraagglutinin. When used in a human, such substances should be suitable forsuch use as a humanized antibody or an enzyme, such as recombinantsialidase of human origin which is non-immunogenic and capable ofcleaving the terminal NeuNAcα6- from the substances of the invention.

It is possible to use the substance according to the invention withother pharmaceutical substance or substances effective against influenzaviruses and achieve more effective pharmaceutical compositions andmethods of treatment, provided that the substances used do notinactivate each other, such as the substance inactivating the substancesaccording to the invention as described above. It is especiallybeneficial to use substances according to the invention together with apharmaceutical neuraminidase (sialidase) inhibitor (Influenza virusneuraminidase inhibitors have been reviewed in The Lancet (2000),355:827-35), which could prevent partial degradation of the substanceaccording to the invention and prevent the viral activity with differentmechanisms.

Furthermore, it is possible to use the substance according to theinvention in the diagnosis of a condition caused by an influenza virusinfection. Diagnostic uses also include the use of the substanceaccording to the invention for typing of the influenza viruses. When thesubstance according to the invention is used for diagnosis or typing, itmay e.g. be included in a probe or a test stick, optionally constitutinga part of a test kit When this probe or test stick is brought intocontact with a sample containing human influenza viruses, the viruseswill bind the probe or test stick and can be thus removed from thesample and further analyzed.

Glycolipid and carbohydrate nomenclature is according to recommendationsby the IUPAC-IUB Commission on Biochemical Nomenclature (Carbohydr. Res.1998, 312, 167; Carbohydr. Res. 1997, 297, 1; Eur. J. Biochem. 1998,257, 29).

It is assumed that Gal, Glc, GlcNAc, and NeuNAc are of theD-configuration, Fuc of the L-configuration, and all the monosaccharideunits in the pyranose form. Glycosidic linkages are shown partly inshorter and partly in longer nomenclature, the linkages of theNeuNAc-residues α3 and α6 mean the same as α2-3 and α2-6, respectively,and β1-3, β1-4, and β1-6 can be shortened as β3, β4, and β6,respectively. Lactosamine refers to N-acetyllactosamine, Galβ1-4GlcNAc,and sialic acid is N-acetylneuraminic acid, NeuNAc. A lactose residue ora lactosamine residue can be derivatized or glycosically conjugated fromposition 1 of Glc/GlcNAc or correspond to a free reducing end of anon-conjugated oligosaccharide chain. In the shorthand nomenclature forfatty acids and bases, the number before the colon refers to the carbonchain length and the number after the colon gives the total number ofdouble bonds in the hydrocarbon chain. Glc/GlcNAc indicates anoligosaccharide continuing either a Glc or a GlcNAc residue in theindicated position and 3/6, 6/3 indicates that the linkage can be eitherto the 3 or the 6 position.

The present invention is further illustrated in examples, which in noway are intended to limit the scope of the invention:

EXAMPLES Abbreviations Used in the Examples

TLC, thin-layer chromatography; C, chloroform; M methanol; MAA, Maackiaamurensis lectin; SNA, Sambucus nigra lectin; MALDI-TOF MS,matrix-assisted laser desorption/ionization time-of-flight massspectrometry; FAB MS, fast atom bombardment mass spectrometry; EI MS,electron ionization mass spectrometry. We use denotations 3s to 8s toindicate migration regions on TLC plates for three- toeight-sugar-containing monosialogangliosides. S-3-PG,sialyl-3-paragloboside (NeuAcα3Galβ4GlcNAcβ-3Galβ4GlcCer); S-6-PG,sialyl-6-paragloboside (NeuAcα6Galβ4GlcNAcβ-3Galβ4GlcCer). Glycolipidand carbohydrate nomenclature, see above.

Materials and Methods

Materials. Horseradish peroxidase (HRP) labeled egg-grown influenza Aviruses (human X-113 reassortant vaccine strain bearing hemagglutininand neuraminidase of A/Texas/36/91, H1N1, and avian virusA/duck/Czechoslovakia/56, H4N6) were prepared as described beforeMatrosovich et al., 1996). Total ganglioside fractions were obtainedfrom the Institute of Medical Biochemistry, Göteborg University, Sweden,and prepared according to Karlsson, 1987. Some fractions were purifiedby phase partition (Folch et al., 1957) before analysis, as indicated inlegends to figures. The human virus-binding subfractions of humanleukocyte gangliosides (FIG. 6) were prepared by preparative thin-layerchromatography (Miller-Podraza et al., 1992) followed by furtherpurification. After separation the fractions were suspended in C/M/H2O,60:30:4.5, by vol, applied to a small (0.25 ml) silica gel column packedin C/M, 2:1, by vol., and the sugar-positive fractions were eluted withC/M H2O, 60:35:8, by vol. Anti-sialyl-Lewis x and CDw65/clone VIM-2monoclonal antibodies were from Seikagakut (Japan) and Dianova GmbH(Germany), respectively, and Maackia amurensis (MAA) and Sambucus nigra(SNA) letins from Boehringer-Mannheim (Germany). Silica gel aluminumplates 60 were purchased from Merck (Germany).

Preparation of leukocytes. Mixtures of human white cells were preparedfrom venous blood of healthy donors. The buffy coats were lysed in 0.8%NH₄Cl (removal of erythrocytes, Fredlund et al., 1988) and centrifugedat 400×g. Fractions used contained from 70% to 85% of polymorphonuclearleukocytes.

Mild periodate oxidation of gangliosides (Veh et al., 1977)

Gangliosides (0.05-0.1 mM) were incubated in 1-2 mM NaIO₄ in 0.05 mMacetate buffer, pH 5.5, for 40 min on ice, after which an excess ofNa₂SO₃ was added. The sample was concentrated by freeze-drying (about5-fold) and reduced with an excess of NaBH₄ at room temperatureovernight. Finally, the sample was dialyzed against distilled water andfreeze dried.

TLC-overlay binding assays. The general overlay technique was previouslydescribed (Karlsson and Strömberg, 1987). Specific applications of thistechnique that we utilized in this study are given below.

Overlay with influenza viruses. Plates with separated glycolipids weretreated with 0.3% polyisobutylmethacrylate (Aldrich Chemical Company,Inc., Milwaukee, USA) in diethyl ether: hexane, 5:1, by vol., for 1 min,dried and incubated in 2% BSA and 0.1% Tween 20 in PBS for 2 h at roomtemperature. The plates were then overlaid with HRP-labeled virussuspension in 0.2% BSA, 0.01% Tween 20 in PBS and incubated as above foradditional 2 h. After washing four times with PBS, the plates werevisualised by incubating at room temperature (in dark) in 0.02% DAB(3,3′-diaminobenzidine tetrahydrochloride, Pierce, Rockford, Ill., USA)in PBS containing 0.03% H₂O₂.

Overlay with antibodies. Overlay with antibodies was performed asdescribed earlier (Miller-Podraza et al. 1997).

Overlay with lectins on membrane blots. Detection of α3- and α6-linkedsialic acids on membrane blots with lectins from Maackia amurensis MAA)and Sambucus nigra (SNA), was performed as described (Johansson et al.,1999).

Mass spectrometry. MALDI-TOF MS was performed on a TofSpec-E (Micromass,UK) mass spectrometer operated in a reflectron mode. The accelerationvoltage was 20 kV and sampling frequency 500 MHz. The matrix was6-aza-2-thiothymine dissolved in CH₃CN. FAB MS was performed on a SX102Amass spectrometer (JEOL) operated in a negative ion mode. The spectrawere produced by Xe atoms (8 kV) using triethanolamine as matrix. EI MSof permethylated glycolipids was performed as described (Breimer et al.,1980) using the same JEOL mass spectrometer.

Example 1 Binding of Human Influenza Virus to Mixtures of HumanGangliosides

FIG. 1 shows binding of human and avian influenza viruses (avian viruseswere used as a control experiment) to reference gangliosides (lane 9)and to mixtures of gangliosides isolated from different human tissues(lanes 1-8). The human influenza virus did not bind in the assayconditions to shorter gangliosides including abundant 5s and 7s species,but displayed a strong and selective binding to some extendedglycolipids of human leukocytes (lane 1 in FIG. 1). There was also aweak binding to slow-moving species of other human tissues, inparticular small intestine and pancreas (lanes 4 and 8). The avian virusbound to a variety of gangliosides in all lanes including two fractionsof reference brain gangliosides, and displayed a preference forNeuAcα3Gal-terminated species as compared with NeuAcα6Gal-terminatedspecies and species with sialic acid as internal branches. As shown inthe figure, there was a binding to NeuAcα3-paragloboside (S-3-PG) andgangliosides GD1a and GT1b (see Table 1 for structures), but not toNeuAcα6-paragloboside (S-6-PG) or gangliosides GM1 or GD1b. Theseresults agree with earlier reports on avian influenza virus bindingspecificities (see refs. in Introduction). The binding to GM3 washowever not observed, although some lanes were overloaded with respectto less complex components. Binding to the fastest-moving band by bothviruses in lane 6 of FIG. 1 was probably unspecific interaction withoverloaded and charged sulfatide.

Example 2 Mild Periodate Oxidation of the Gangliosides

To exclude binding to sulfated glycolipids in other lanes we used mildperiodate oxidation and reduction which shortens specifically the sialicacid glycerol tail in gangliosides by one or two carbon atoms (Veh etal., 1977). Binding of influenza viruses to sulfated galactosylceramidewas previously reported by Suzuki et al., 1996. The mild oxidationeliminated in our studies completely (human influenza) or almostcompletely (avian virus) binding to leukocyte glycolipids, as shown inFIG. 2. The result confirms the specific importance of the sialic acidfor the virus attachment, and agrees with previous studies on binding ofinfluenza viruses to chemically modified carbohydrates (Suttajit andWinzler, 1971; Matrosovich et al., 1991).

The oxidized and reduced gangliosides were tested by FAB MS and by EI MSafter permethylation. In FAB MS spectra the pseudomolecular ions [M-H]⁻seen clearly for 3s, 5s and 7s gangliosides were reduced by 30 or 60(±1) mass units, see FIG. 3. Thus, the main ions in FIG. 3A at m/z1151.7(GM3, d18:1-16:0), 1517.2 (SPG, d18:1-16:0) and 1882.7 (7s,d18:1-16:0) were replaced after oxidation and reduction in FIG. 3B byions at 1091.1 and 1121.1, 1456.4 and 1486.4, and 1821.9 and 1851.8,respectively. In EI MS spectra the NeuAc fragment ions at m/z 376 and344, were replaced after oxidation and reduction by m/z at 332 and 300,and 288 and 256 (not shown). Degradation of the core chains was notobserved.

Example 3 Analysis of the Human Influenza Receptor Glycolipids byAntibodies

To test if the binding of the human virus was to the monofucosylatedsialyl-Lewis x or VIM-2-active saccharides as reported earlier (Suzuki,1994; Müthing, 1996), we used overlay of TLC plates with antibodieswhich react with these structures (FIG. 4). The polar solvent (A)allowed clear separation of VIM-2- and virus-positive fractions (FIG.4A, lanes VIM and Virus), at least within the less complex region. Therewas an overlapping of anti-sialyl-Lewis x and human virus bindings(FIGS. 4A and 4B, lanes SLX and Virus), but the patterns were notidentical, and there was no recognition by the virus of less complexsialyl-Lewis x-positive molecules.

Example 4 Characterization of the Human Influenza Receptor Glycolipidsby Lectins

FIG. 5 shows binding of MAA and SNA (lectins specific for α3- andα6-linked NeuAc, respectively) to leukocyte gangliosides in comparisonwith binding by human influenza virus. There was a co-migration of themore complex virus- and SNA-positive fractions, however, there was nobinding of the virus to the less complex SNA-positive bands. The latterwere earlier identified as 5s and 7s gangliosides with 6-linked NeuAc(Johansson and Miller-Podraza, 1998). MAA, which binds specifically toNeuAcα3-containing carbohydrate chains of the neolacto series (Knibbs etal., 1991; Johansson et al., 1999), displayed a completely differentpattern of binding than the virus. Binding of the human virus to minorNeuAcα6-containing species were reproduced after ganglioside separationin a second TLC solvent (not shown). The weak binding of the virus seenin lane 3 of FIG. 5 could represent cross reaction with braingangliosides. However this binding was not reproducible (see FIG. 1).

In an attempt to identify sequences that display efficient binding ofhuman virus, gangliosides from human leukocytes were separated bypreparative TLC and tested again for the binding (FIG. 6). The mostcomplex fractions were not analysed in this way because of inadequateamounts and poor separation. Two fractions were shown to contain activecomponents, see Fr. 5 and Fr. 6 in the figure. Tests in differentchromatographic systems (TLSC not shown) revealed however that the maincomponents in these subfractions were inactive and that binding was tosome minor overlapping fractions. This agrees with the fact that thebinding was to slowly migrating NeuAcα6-containing species (FIG. 5),which have earlier been detected by SNA lectin (Johansson andMiller-Podraza, 1998). They occur in human leukocytes in very smallamounts and so far only NeuAcα3-containing structures were detected bychemical methods among more complex gangliosides isolated from thissource (Müthing et al., 1996; Stroud et al., 1995; Stroud et al., 1996).The detection level on TLC plates by the human virus was 40-80 pmol inrelation to the total ganglioside mixture, and apparently much lower forthe active species, indicating that the binding was highly efficient(see binding to trace fractions in lane 6 of FIG. 6)

Example 5 Mass Spectrometry of Glycolipid Fractions Containing Receptorsfor Human Influenza Viruses

The most active isolated fraction (Fr. 6 of FIG. 6) was shown byMALDI-TOF mass spectrometry to contain complex gangliosides with 8-11monosaccharides per mol of ceramide (FIG. 7 and Table 2). Fragmentationpattern seen in FAB MS analysis confirmed the presence ofoligosaccharide chains with repeated HexHexNAc units (spectra notshown). The most abundant molecular ions corresponded toNeuAc₁Fuc₁Hex₅HexNAc₃Cer at m/z 2394.0 and NeuAc₁Hex₅HexNAc₃Cer at2248.7. As mentioned, the main components were excluded as bindingmolecules by TLC in different solvent systems. The most probablecandidates of the active species were therefore minor disialylated ordifucosylated gangliosides with 2 or 3 lactosamine units.Monofucosylated gangliosides NeuAc₁Fuc₁Hex₄HexNAc₂Cer, (8s), (m/z at2030.4 in FIG. 7) with two N-acetyllactosamine units are less likely asbinding molecules, as judged from chromatographic mobility and bindingtests, see FIGS. 1, 4B and 6, (8s region). In Fr. 5 of FIG. 6 the main(non-active) component was NeuAc₁Hex₅HexNAc₃Cer.

Example 6 Analysis of Data from Mass Spectrometry and TLC-Experiments

The direct characterization of the exact binding structures was notpossible because of the low amount of the material and presence ofseveral active species. However, we have excluded sialyl-Lewis x andVIM-2-active saccharides (Table 1, FIG. 4) as the binding sequences. Wehave also shown using SNA lectin that the binding was to some minorNeuAcα6-containing species (FIG. 5). In addition, we have demonstrated,that the binding was dependent on the presence of the unchanged glycerolsialic acid tail (FIGS. 2 and 3). Our results therefore do not supportearlier suggestions on a preferential binding of human influenza Aviruses to sialyl-Lewis x (Suzuki, 1994) or VIM-2-active structures(Suzuki, 1994; Müthing, 1996). This discrepancy may be caused bydifferences in virus stains. The cited authors used influenza virusesA/PR/8/34 (H1N1) and the reassortant virus strain X-31, which bear HAand NA genes of A/Aichi/2/28 (H3N2), while we studied human influenzavirus X-113 (HA and NA genes of A/Texas/36/91, H1N1). PR/8/34 and X-31are known to have a higher affinity to NeuAcα3-containing receptors thanmore recently circulated influenza A viruses (Rogers and D'Souza, 1989;Matrosovich et al., 1997), and this may explain strong interaction ofPR/8/34 and X-31 with sialyl-Lewis x and VIM-2 structures and no bindingto these structures in our tests. There was an overlapping binding byanti-sialyl-Lewis x antibody and the human virus in our studies (FIG.4). However, the overall patterns were not identical and there was nointeraction of the virus with less complex sialyl-Lewis x gangliosidesreported to be present in human leukocytes (Müthing et al., 1996). Wehave detected by FAB MS in fractions 1 through 3 (FIG. 6) increasingamounts of 8s gangliosides with a potential sialyl-Lewis x compositionof Fuc₁NeuAc₁Hex₄HexNAc₂Cer with various ceramides (m/z at 2028.7 and2138.4, not shown). These gangliosides migrated in the 7s regionoverlapping with more abundant non-fucosylated NeuAc₁Hex₄HexNAc₁Cerfractions. Of importance is a binding in this region ofanti-sialyl-Lewis x antibody, but not of either VIM-2 antibody or virus(FIG. 4). Structural microheterogeneity associated with ceramide partsand NeuAcα3/α6 substitutions may explain the overlapping migration ofdifferent gangliosides and the complex multi-band patterns in lanes SLXof FIG. 4. The cross-binding to other fucosylated structures (Stroud etal., 1995) of the lower TLC regions, may also contribute to thesecomplex patterns.

We have excluded that the main components of fractions 5 and 6 in FIG. 6are binding molecules by TLC analysis in different solvent systems andoverlay tests. Table 2 lists (in bold) candidates of active specieswhich we could detect by MALDI-TOF MS. Of importance for the bindingcould be oligosialylation and/or repeated fucose branches, as judgedfrom the presence of disialylated molecules and/or difucosylatedmolecules in the mixture (difference between masses of 2Fuc and 1NeuAcis only 1.03 amu). Like the oligosialylated species the monosialylatedglycolipid may also be branched by β6-linked lactosamine unit, molecularmass of such minor species would overlap the major non-active glycolipidwith [M-H]⁻ molecular mass at 2248.5. Also length of the sugar should beconsidered as an important factor, since only complex gangliosides werebinding. The extended carbohydrate chains may serve as spacers whichreduce steric hindrance to recognition by viral hemagglutinins. It hasbeen shown, that glycosylation of viral HAs in the vicinity of thereceptor binding sites may decrease the virus binding to target cellsand immobilized receptors (Matrosovich et al., 1997; Gambaryan et al.,1998; Ohuchi et al., 1997). The impaired accessibility of the receptorbinding pocket could explain why we did not see binding of the humaninfluenza to less complex gangliosides like GM3 or SPG. In fact, thehemagglutinin of X-113 reassortant human virus, although not yetsequenced, is likely to contain glycans at Asn₁₂₉ and Asn₁₆₃ close tothe tip of the HA globular head, similar to the HAs of othercontemporary H1N1 human viruses, for which sequences are available. Incontrast, the avian virus strain A/duck/Czechoslovakia/56 (H4N6) lackscarbohydrates in this portion of the HA (Matrosovich et al., 1999). AlsoMüthing (Müthing, 1996) emphasized a stronger binding to longerfucosylated species (sialyl-Lewis x- and VIM-2-active species) comparedto 5s and 7s gangliosides using X-31 (H3N2) influenza A strain.

In our studies there was however no binding at all to 5sNeuAcα6-containing SPG nor to its 7s homologue, although the interactionof the human virus with some selected complex species was very strong(for characterization of 5s and 7s gangliosides of human leukocytes seeJohansson and Miller-Podraza, 1998). Technical assay reasons for thisunusual binding are unlikely, since the avian virus bound to S-3-PG andto other well defined common gangliosides under the same experimentalconditions (FIG. 1). It is reasonable to assume, that both length andstructural features of the receptor chain contributed to this result.Gangliosides with branched N-acetyl-lactosamine chains and NeuAc on morethan one arm should be considered as highly efficient binding molecules.Polyvalency has earlier been shown as an important factor enhancingbinding affinity of influenza virus to synthetic sialylated compounds(Mammen et al., 1995), and branched polyglycosylceramides were veryeffective receptors for human influenza A and B viruses (Matrosovich etal., 1996). The calculated molecular masses of the disialylated specieshaving similar ceramide structures as previous reported from the humanleukocyte gangliosides (Stroud et al., 1996b; Müthing, 1996) matchedclosely to the experimental data in Table 2. Branched α3 monosialylatedpolylactosamine ganglioside has been described from human gangliosides(Stroud et al, 1996b). Human leukocytes are known to contain neolactoglycolipids with repeated fucose residues as Fucα3GlcNAc units (Stroudet al., 1995; Stroud et al., 1996; Müthing, 1996), correspondingmolecular weights are also seen in our analyses, see Table 2. Fucose maypossibly interact with the hydrophobic methyl group with spots outsidethe NeuAc binding site of the viral HA. In fact, synthetic NeuAcanalogues with hydrophobic neighbouring groups have been shown tointeract with hydrophobic patches adjoining the receptor binding site ofinfluenza virus A hemagglutinin, considerably improving affinity(Watowich et al., 1994).

TABLE 1 Carbohydrate and glycolipid structures discussed in this paperNeuAcα3Galβ4GlcβCer GM3, 3s gangliosideGalβ3GalNAcβ4(NeuAcα3)Galβ4GlcβCer GM1NeuAcα3Galβ3GalNAcβ4(NeuAcα3)Galβ4GlcβCer GD1aGalβ3GalNAcβ4(NeuAcα8NeuAcα3)Galβ4GlcβCer GD1bNeuAcα3Galβ3GalNAcβ4(NeuAcα8NeuAcα3)Galβ4GlcβCer GT1bNeuAcα3Galβ4GlcNAcβ3Galβ4GlcβCer S-3-PG, NeuAc-3-paragloboside,sialyl-3-paragloboside, 5s ganglioside NeuAcα6Galβ4GlcNAcβ3Galβ4GlcβCerS-6-PG, NeuAc-6-paraglobiside, sialyl-6-paragloboside, 5s gangliosideNeuAcα3Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4GlcβCer 7-sugar NeuAc-3-neolactoganglioside, 7s gangliosideNeuAcα6Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4GlcβCer 7-sugar NeuAc-6-neolactoganglioside, 7s ganglioside NeuAcα3Galβ4(Fucα3)GlcNAcβ- Sialyl-Lewis xepitope NeuAcα3Galβ4GlcNAcβ3Galβ4(Fucα3)GlcNAcβ- “VIM-2” epitope(epitope reacting with CDw65/clone VIM-2 monoclonal antibody)

TABLE 2 Ganglioside composition of fraction 6 (Fr. 6 in FIG. 6) based onMALDI-TOF-MS mass spectrometry. The spectrum is shown in FIG. 7. Themost probable binding components (as judged from combined results) arein bold. M-H M-H Most probable compositions (listed Observed CalculatedΔM according to decreasing abundance) 2394.0 2394.6 −0.6NeuAc₁Fuc₁Hex₅HexNAc₃Cer (d18:1-16:0) 2248.7 2248.5 +0.2NeuAc₁Hex₅HexNAc₃Cer (d18:1-16:0) 2504.4 2504.8 −0.4NeuAc₁Fuc₁Hex₅HexNAc₃Cer (d18:1-24:1) 2173.7 2174.4 −0.7 NeuAc ₂ Hex ₄HexNAc ₂ Cer (d18:1, 16:0) and/or 2173.4 +0.3 NeuAc ₁ Fuc ₂ Hex ₄ HexNAc₂ Cer (d18:1- 16:1) 2649.3 2649.9 −0.6 NeuAc ₂ Hex ₅ HexNAc ₃ Cer(d18:1-24:1) and/or 2651.0 −1.7 NeuAc ₁ Fuc ₂ Hex ₅ HexNAc ₃ Cer (d18:1-24:1) and/or 2649.0 +0.3 NeuAc ₁ Fuc ₂ Hex ₅ HexNAc 3 Cer (d18:1- 24:2)2030.4 2029.3 +1.1 NeuAc₁Fuc₁Hex₄HexNAc₂Cer (d18:1-16:0)

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1. A method for the treatment of a condition caused by human influenza virus, said method comprising administering a pharmaceutically effective amount of a substance to a patient in need thereof, wherein said substance comprises at least one oligosaccharide chain, said oligosaccharide chain comprising a terminal NeuNAcα6 linked to (a) a branched polylactosamine type structure consisting of at least three lactosamine residues glycosidically linked to each other, wherein: the structure optionally contains one or more additional NeuNAcα6 residues at a terminal position in a branch; and/or (b) a branched structure consisting of two lactosamine and one lactose residue glycosidically linked to each other, wherein: the structure optionally contains one or more additional NeuNAcα6 residues at a terminal position of the branch, and wherein said substance contains two NeuNAcα6 residues in a branched structure.
 2. A method for the treatment of a condition caused by human influenza virus, said method comprising administering a pharmaceutically effective amount of a substance to a patient in need thereof, wherein said substance comprises at least one oligosaccharide chain, said oligosaccharide chain comprising a terminal NeuNAcα6 linked to (a) a linear polylactosamine type structure consisting of at least three lactosamine residues glycosidically linked to each other, wherein: the structure contains optionally one or two fucose residues, each of which is α3-linked to a GlcNAc-residue of a lactosamine residue to which the terminal NeuNAc is not α6-linked; and/or (b) a linear or branched structure consisting of two lactosamine and one lactose residue glycosidically linked to each other, wherein: the structure additionally contains one or two fucose residues, each of which is α3-linked to GlcNAc-residue of a lactosamine residue to which the terminal NeuNAc is not α6-linked; or α3-linked to Glc-residue of a lactose residue to which NeuNAc is not α6-linked; and wherein said substance contains one fucose residue in the linear structure consisting of at least three lactosamine residues, or two fucose residues in the linear structure with two lactosamine residues and one lactose residue.
 3. The method according to claim 1 or 2, wherein said oligosaccharide chain comprises a terminal NeuNAcα6 linked to three lactosamine residues.
 4. The method according to claim 1 or 2, wherein the condition due to the presence of human influenza virus is a primary infection by an influenza virus in the respiratory tract.
 5. The method according to claim 1 or 2, wherein the condition due to the presence of human influenza virus is a secondary bacterial infection.
 6. The method according to claim 1 or 2, wherein a pharmaceutically effective amount of one or more additional substances effective against influenza viruses is administered.
 7. The method according to claim 1 or 2, wherein said additional substance is a neuraminidase inhibitor.
 8. A method for the treatment of a condition caused by human influenza virus, said method comprising administering a pharmaceutically effective amount of a substance to a patient in need thereof, wherein said substance comprises at least one oligosaccharide chain, said oligosaccharide chain comprising a terminal NeuNAcα6 linked to (a) a linear or branched polylactosamine type structure consisting of at least three lactosamine residues glycosidically linked to each other, wherein: when the structure is a linear structure, the structure contains optionally one or two fucose residues, each of which is α3-linked to a GlcNAc-residue of a lactosamine residue to which the terminal NeuNAc is not α6-linked; or when the structure is a branched structure, the structure optionally contains one or more additional NeuNAcα6 residues at a terminal position in a branch, and/or (b) a linear or branched structure consisting of two lactosamine and one lactose residue glycosidically linked to each other, wherein: when the structure is said linear type structure, the structure additionally contains one or two fucose residues, each of which is α3-linked to GlcNAc-residue of a lactosamine residue to which the terminal NeuNAc is not α6-linked; or α3-linked to Glc-residue of a lactose residue to which NeuNAc is not α6-linked; or when the structure is said branched structure, the structure optionally contains one or more additional NeuNAcα6 residues at a terminal position of the branch, wherein said substance contains an oligosaccharide structure of NeuNAcα6Galβ4GlcNAcβ3(NeuNAcα3/6Galβ4GlcNAcβ6)Galβ4GlcNAc/Glcβ1-, NeuNAcα6Galβ4GlcNAcβ3(NeuNAcα6Galβ4GlcNAcβ6)Galβ4GlcNAc/Glcβ1-, NeuNAcα6Galβ4GlcNAcβ3(Galβ4GlcNAcβ6)Galβ4GlcNAc/Glcβ1-, NeuNAcα6Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4(Fucα3)GlcNAcβ1-, or NeuNAcα6Galβ4GlcNAcβ3Galβ4(Fucα3)GlcNAcβ3Galβ4GlcNAcβ1.
 9. A method for the treatment of a condition caused by human influenza virus, said method comprising administering a pharmaceutically effective amount of a substance to a patient in need thereof, wherein said substance comprises at least one oligosaccharide chain, said oligosaccharide chain comprising a terminal NeuNAcα6 linked to (a) a linear or branched polylactosamine type structure consisting of at least three lactosamine residues glycosidically linked to each other, wherein: when the structure is a linear structure, the structure contains optionally one or two fucose residues, each of which is α3-linked to a GlcNAc-residue of a lactosamine residue to which the terminal NeuNAc is not α6-linked; or when the structure is a branched structure, the structure optionally contains one or more additional NeuNAcα6 residues at a terminal position in a branch, and/or (b) a linear or branched structure consisting of two lactosamine and one lactose residue glycosidically linked to each other, wherein: when the structure is said linear type structure, the structure additionally contains one or two fucose residues, each of which is α3-linked to GlcNAc-residue of a lactosamine residue to which the terminal NeuNAc is not α6-linked; or α3-linked to Glc-residue of a lactose residue to which NeuNAc is not α6-linked; or when the structure is said branched structure, the structure optionally contains one or more additional NeuNAcα6 residues at a terminal position of the branch, wherein said oligosaccharide is a part of a glycolipid.
 10. A method for the treatment of a condition caused by human influenza virus, said method comprising pharmaceutically effective amount of a substance to a patient in need thereof, wherein said substance comprises at least one oligosaccharide chain, said oligosaccharide chain comprising a terminal NeuNAcα6 linked to (a) a linear or branched polylactosamine type structure consisting of at least three lactosamine residues glycosidically linked to each other, wherein: when the structure is a linear structure, the structure contains optionally one or two fucose residues, each of which is α3-linked to a GlcNAc-residue of a lactosamine residue to which the terminal NeuNAc is not α6-linked; or when the structure is a branched structure, the structure optionally contains one or more additional NeuNAcα6 residues at a terminal position in a branch, and/or (b) a linear or branched structure consisting of two lactosamine and one lactose residue glycosidically linked to each other, wherein: when the structure is said linear type structure, the structure additionally contains one or two fucose residues, each of which is α3-linked to GlcNAc-residue of a lactosamine residue to which the terminal NeuNAc is not α6-linked; or α3-linked to Glc-residue of a lactose residue to which NeuNAc is not α6-linked; or when the structure is said branched structure, the structure optionally contains one or more additional NeuNAcα6 residues at a terminal position of the branch, said substance comprising at least two oligosaccharide chains. 